Use of lactic acid bacteria for reducing dental caries and bacteria causing dental caries

ABSTRACT

Strains of  Lactobacillus  that have been selected for their capability of reducing the number of  Streptococcus mutans  in the mouth of mammals through inhibiting activity in combination with good binding to the oral mucins and dental plaque, thereby preventing, reducing or treating dental caries, and products derived from said strains, including agents for treatment or prophylaxis of caries for administration to humans.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to use of a method for screening nonpathogenicanti-cariogenic strains, and products and methods using such strains fortreatment and prophylaxis of dental caries caused by oral bacteria suchas Streptococcus mutans, and other caries-causing pathogens.

2. Description of the Related Art

The oral cavity of humans and other mammals contains many differentspecies of bacteria, including a number of different species ofLactobacillus. Caries is a disease caused by bacteria. In 1890, Millerin “Chemico-Parasitic Theory” presented the hypothesis that caries iscaused by oral bacteria producing acids from digestive carbohydrates,which dissolve the hydroxyhepatite of the teeth. It was later confirmedin gnotobiotic rats, for example, that normal oral bacterial flora, areinvolved, primarily of the mutans streptococci group, and also that thelactobacilli group is involved. These “acidogenic” species resident inthe oral cavity are associated with the presence and onset of dentalcaries (Locsche W J, Microbiolog Rev., 1986: 50: 353-380). There areseven bacterial species within the group mutans streptococci, withStreptococci mutans (serotype c,e,f) being found in 90% of all humanisolates (Linder L., Oral Mikrobiologi 1996, ISBN 91-7205-037-3). Thereis abundant evidence that the initiation of caries requires a relativelyhigh proportion of S. mutans within dental plaque. These bacteria adherewell to the tooth surface, produce higher amounts of acid from sugarsthan other bacterial types, can survive better than other bacteria in anacid environment, and produce extracellular polysaccharides fromsucrose. When the proportion of S. mutans in plaque is high (in therange of 2-10%), a patient is at high risk for caries. When theproportion is low (less than 0.1%), the patient is at low risk. Becausethey are more acid tolerant than other bacteria, acid conditions withinplaque favor the survival and reproduction of mutans streptococci. Twoother types of bacteria are also associated with the progression ofcaries through dentin. These are several species of Lactobacillus, andActinomyces viscosus. These bacteria are also highly acidogenic andsurvive well in acid conditions. The involvement of Lactobacillus indental caries has been established (Smith et al., Microbios 105: 77-85,2001). In fact, estimation of the lactobacillus counts in saliva, inaddition to the estimation of mutans streptococci counts, usingdifferent selective media or other techniques, has been used for manyyears as a “caries test” and to attempt to identify groups at high riskfor caries. Thus, Lactobacillus strains, some isolated from human dentalplaque, may be highly cariogenic (Fitzgerald et al., J. Dent. Res. 60:919-926, 1981.

For a bacteria to be a primary pathogen in the formation of dentalcaries it is required that it have a combination of several of therequired characteristics (Linder, 1996): ability to adhere and colonizeon the teeth surface; ability to accumulate in large numbers on alimited surface of the teeth; ability to quickly produce acid fromcarbohydrates found in foods; and ability to continue acid productioneven under low pH in the dental plaque.

Dietary sucrose changes both the thickness and the chemical nature ofplaque. Mutans streptococci and some other plaque bacteria use themonosaccharide components (glucose and fructose) and the energy of thedisaccharide bond of sucrose to assemble extracellular polysaccharides.These increase the thickness of plaque substantially, and also changethe chemical nature of its extracellular space from liquid to gel. Thegel limits movement of some ions. Thick gel-plaque allows thedevelopment of an acid environment against the tooth surface, protectedfrom salivary buffering. Plaque which has not had contact with sucroseis both thinner and better buffered. A diet with a high proportion ofsucrose therefore increases caries risk. Thicker plaque occurs in pitsand fissures and, in patients with poor oral hygiene, near the gingivalmargin.

Given this concept of the nature of the disease, it is clear thatprevention and treatment of dental caries requires hindering the effectsof S. mutans, for example, through dietary change as means of reducingthe substrate for the bacteria, to reinforce the surface structure ofthe teeth or reduction of the number of S. mutans bacteria. Thus,treatments that have been tried include: efforts at changing themicroflora, using agents such as topical chlorhexidine and topicalfluoride; reducing the amount of dietary sucrose, by dietary change andsubstitution for sweeteners more difficult to metabolize by S. mutans,such as Sorbitol, Aspartan, Xylitol; decreasing the frequency of eating,by dietary choice; adding fluoride, particularly through dailyapplication during tooth brushing; and increasing salivary flow, usingmechanical stimulation during vigorous chewing to enhance flow, bychanging drugs which reduce flow, or by using drugs to enhance flow.Different approaches has been evaluated for preventing dental caries,for example, one composition uses a lytic enzyme produced by abacteriophage specific for Streptococcus mutans (U.S. Pat. No. 6,399,098of Fischetti et al.). Also, a strain of Lactobacillus zeae has beenmodified through genetical engineering to produce an antibody on itssurface to neutralize the detrimental streptococcal bacteria,(Hammarstrom L., July 2002 issue of Nature Biotechnology), however thisapproach with genetically modified organisms faces an unknown safetyapproval situation.

In addition, one strain of Lactobacillus rhamnosus (strain GG) has beenpromoted as a probiotic method of reducing Streptococcus sabrinus andmutans streptococci generally (Nase et al., Caries Res. 35: 412-420,2001). Further work showed that use of this strain as a starter infermenting milk did not influence the titer of antibodies against humancariogenic bacteria that were present in the milk (Wei et al., OralMicrobio. & Immunol. 17: 9-15, 2002. L. rhamnosus GG differs from L.reuteri in many ways, including fermentation characteristics andisolation source. Other microorganisms that have been found to haveinhibitory activity against the formation of dental plaque includeEnterococcus, Lactobacillus acidophilus V20, and Lactobacillus lactis1370 (Oh, U.S. Pat. No. 6,036,952). In order to inhibit S. mutans, otherwork has been done using so called “competitive exclusion” concepts. Forexample, L. reuteri strain ATCC 55730 has been shown to inhibit S.mutans (Nikawa H. et al, News release by Hiroshima University Jul. 11,2002). A tablet product which is on the market in Japan called LS 1,containing a strain of Lactobacillus salivarius (LS 1) (by Frente Ltd.Japan) is claimed to inhibit S. mutans.

Strains of a wide variety of Lactobacillus species, includingLactobacillus reuteri, have been used in probiotic formulations.Lactobacillus reuteri is one of the naturally occurring inhabitants ofthe gastrointestinal tract of animals, and is routinely found in theintestines, and occasionally in the birth channel, breast milk and mouthof healthy animals, including humans. It is known to have antibacterialactivity. See, for example, U.S. Pat. Nos. 5,439,678, 5,458,875,5,534,253, 5,837,238, and 5,849,289. When L. reuteri cells are grownunder anaerobic conditions in the presence of glycerol, they produce theantimicrobial substance known as reuterin (β-hydroxy-propionaldehyde).Other antimicrobial substances beside the traditional organic acids havealso been reported such as “Reutericyclin” (Holtzel, A. et al.Angewandte Chemie International Edition 39, 2766-2768, 2000) and “PCA(pyroglutamic acid)” (Yang, Z. Dissertation, Univ. of Helsinki, March2000). Lactobacilli, including L. reuteri, are also well known to havethe ability to inhibit other organisms such as S. mutans through localcompetition of nutrients and other metabolic interactions.Immunomodulating and anti-inflammatory activity has also been associatedwith L. reuteri. Mucin binding proteins of L. reuteri have been isolatedand described. See, for example, U.S. Pat. No. 6,100,388.

Lactobacillus strains have been reported to adhere to various cell linesand host mucus. This has been speculated to be important for probioticactivity and is derived from the concept of virulence factors inpathogenic bacteria, where vast arrays of such interactions have beendiscovered during the last decades (Klemm, P. and Schembri, M. A. (2000)Bacterial adhesins: function and structure. Int. J. Med. Microbiol. 290,27-35.)

While the possibility of effective antibacterial activity by L. reuteriis known, and certain binding characteristics of L. reuteri such asmucin binding are known, and S. mutans inhibiting effects of L. reuteristrain ATCC 55730 and Lactobacillus GG ATCC 53103 are also known, it wasnot previously known that substantial differences existed betweenlactobacilli strains in their ability to reduce the number ofStreptococci mutans in the oral cavity and thereby caries, as aconsequence of both inhibiting effect and binding activity, nor thatsuch strains could be selected.

It is therefore an object of the invention to provide better strains ofLactobacillus which have been selected for their capability of reducethe number of S. mutans in the mouth through antimicrobial activity incombination with good binding to the oral mucins and dental plaque andthereby prevent, reduce or treat dental caries. It is a further objectof the invention to provide products containing said strains, includingagents for prophylaxis or treatment of caries associated with S. mutansfor administration to humans.

Other objects and advantages will be more fully apparent from thefollowing disclosure and appended claims.

SUMMARY OF THE INVENTION

The invention herein comprises strains of Lactobacillus that have beenselected for their capability of reducing the number of Streptococcusmutans in the mouth of mammals through inhibiting activity incombination with good binding to the oral mucins and dental plaque,thereby preventing, reducing or treating dental caries, products derivedfrom said strains, including agents for treatment or prophylaxis ofcaries for administration to humans, and a method for producing theseproducts.

Other objects and features of the inventions will be more fully apparentfrom the following disclosure and appended claims.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The present invention provides a product, for inhibiting the growth andactivity of dental caries bacteria, comprising cells of at least oneselected strain of Lactobacillus with both good antimicrobial activityagainst Streptococci mutans combined with good binding to the oralmucins and dental plaque and thereby prevent, reduce or treat dentalcaries. Such strains include L. reuteri CF2-7F (ATTC deposited on Jan.29, 2003, number to be included herein when assigned) and L. reuteriMF2-3 (ATTC deposited on Jan. 29, 2003, number to be included hereinwhen assigned).

In addition to their ability to reduce the number of Streptococci mutansin the oral cavity, the other criterion for Lactobacillus strainselection in this invention is the ability of the strain to adhere tothe host mucins. Indeed, it has been shown that lactobacilli do adhereto mucosal surfaces and components thereof. Working with adhesion oflactobacilli to mucus revealed that many strains apparently lack theability to bind mucus material in vitro. Since many of these non-binderswere isolated from mucosal surfaces, it could be assumed that theenvironment of growth could affect the adhesion properties of thebacteria.

In the selection method used herein, the adhesion characteristics havebeen examined by partly mimicking the intestinal environment byincluding mucin in the bacterial growth medium. Thus, as discussed inthe Examples, the method of selection of the invention comprises: 1)evaluation of the inhibiting effect of S. mutans by Lactobacillusstrains; and 2) evaluation of mucin binding effect by Lactobacillus.

The product of the invention can be any product for placement in themouth as a preventative or treatment for dental caries, or fornutritional or breath purpose, such as food products, dental treatmentproducts such as mouthwashes or other specified health product, chewinggum, and the like. Food products lending themselves particularly to usein the invention include milk-containing products such as yogurt, andalso juices, drinks and the like. The dental treatment products that maybe used in the invention include toothpastes, liquid tooth cleansers,mouthwashes, anti-halitosis products, and the like.

The concentration of selected Lactobacillus cells needed foreffectiveness of a product of the invention depends on the type of foodand the amount of food to be ingested (or the time of use in the mouthof a non-food dental treatment product), but it is usually preferable tohave about 10⁶-10⁷ CFU (colony-forming units) or more per gram of aproduct. Amounts up to about 10¹⁰-10¹¹ CFU are possible and can be usedto increase efficacy without adversely affecting the product'sorganoleptic characteristics (its flavor or smell). When the product isyogurt or other lactic acid fermentation product, the lactic acidfermentation strain(s) used to produce the product would preferably bestandard cultures for this particular purpose, and the anti-cariogenicbacteria of the invention may be added either before or after thefermentation of the product at a level of about 10⁶-10⁷ CFU per ml ofyogurt or more as discussed above.

Preferably the product of the invention does not contain otherantibacterial components, at least none that inhibit or kill selectedLactobacillus strain(s) or interfere with its anti-cariogenic activity.

The strain(s) of Lactobacillus can be an additive mixed into theingredients or kneaded into or coated on the product by means known inthe art for formulation of products of that type. If preparation of theselected food or other product of the invention requires a heating step,the Lactobacillus strain(s) should be added after the heating. Once theselected Lactobacillus cells are in the product, it is preferred not toheat the product to 60-70 degrees C. or above for a longer period oftime.

The features of the present invention will be more clearly understood byreference to the following examples, which are not to be construed aslimiting the invention.

EXAMPLE 1 Method of Selection of Strains

The selection of the Lactobacillus strains to be used according to thisinvention can be done in the following two step manner:

a) Evaluation of Inhibiting Effect of S. mutans by Lactobacillus Strains

An example of a strain to use to measure the inhibitory effect isStreptococcus mutans, ATCC25175 (available from The American TypeCulture Collection, Manassas, Va., USA). The isolate is grown intrypticase soy broth (Difco, Detroit, USA) supplemented with 0.5% yeastextract (Difco) (TSBY). The cells are harvested during the exponentialgrowth phase by centrifugation at 1000×g, washed twice with PBS andresuspended in the same buffer. The cell suspensions are subjected to alow-intensity ultrasonic device to disperse bacterial aggregates.

The test Lactobacillus strain is grown in brain-heart infusion broth(Difco), harvested during the exponential growth phase by centrifugationat 1000×g, washed twice with phosphate buffered saline (PBS; pH 6.8) andre-suspended in the same buffer.

The optical densities of the bacterial suspensions are measured in a 1.0ml cuvette with a 1 cm light path, and the suspensions is adjusted to afinal concentration of 1.0×10⁸ CFU (colony forming unit)/ml.

The inhibitory assay is conducted as follows, the suspension of S.mutans and the suspension of Lactobacillus are mixed in the ratios of100-0, 75-25, 50-50 and 25-75 in sterile centrifugation tube (totalvolume 100 μl), added the BHI broth up to 10 ml, vortex mixed for tenseconds and incubated for 90 min at 37° C. with gentle shaking. As acontrol, the suspension of S. mutans is mixed with an equal volume ofPBS in the control tubes (free of Lactobacillus). Afterwards eachsuspension is washed by centrifugation at 1000×g, washed twice with PBS,and plated on MS agar to determine the CFU count of S. mutans. The %survival of S. mutans is obtained from following formula.${\%\quad{survival}\quad{of}\quad{S.\quad{mutans}}} = {\frac{{CFU}\quad{of}\quad{S.\quad{mutans}}\quad{incubated}\quad{with}\quad{Lactobacillus}}{{CFU}\quad{of}\quad{S.\quad{mutans}}\quad{incubated}\quad{with}\quad{PBS}} \times 100}$

The assay should be carried out with minimum triplicate samples. All thenumerical data obtained should be statistically analyzed.

b) Evaluation of Mucin Binding Effect by Lactobacillus

Mucine from porcine stomach (Sigma, St. Louis, Mo., USA) is suspended incarbonate buffer at pH 9.7 at a concentration of 0.1 mg/ml. 200 μl ofthe solution is pipetted into microtiter wells and is left for coatingat 37° C. for approximately 3 hours. The wells are blocked by theaddition of 200 μl PBS 1% Tween20 at room temperature for 1 hour andwashed 3 times with PBS 0.5% Tween20 (PBST). The Lactobacillusevaluation strains are grown overnight at 37° C. in MRS brothsupplemented with 0.01% pig gastric mucin (Sigma, M1778) to mimic thegrowth conditions in vivo, then washed and re-suspended in PBST. Opticaldensity (OD) of the bacterial cells is measured at 600 nm in a BeckmanDU650 spectrophotometer and adjusted to OD 0.5.100 μl bacterialsuspension is added to each well and incubated overnight at 4° C. Thewells are washed with PBST, and binding is examined with an invertedmicroscope. The buffer is poured off and, after the wells had dried,OD405 measured in an ELISA plate reader. The results of the binding isscored on a scale from 0 to 3, where 0 is no binding and 3 is strongbinding to the mucin.

The Lactobacillus strains showing best results in both inhibiting of S.mutans as well as binding to mucin, according to the assays areselected.

EXAMPLE 2 Selection of Strains

-   1. L. reuteri SD2112 (ATCC 55730)-   2. L. reuteri DSM 20016 (DSM 20016)-   3. L. reuteri MM2-3 (ATCC PTA-4659)-   4. L. reuteri CF2-7F (ATTC number to be included herein when    assigned)-   5. L. reuteri MF2-3 (ATTC number to be included herein when    assigned)-   6. L. reuteri MF14-C (Culture collection of Biogaia AB, Raleigh    N.C., USA)-   7. L. reuteri MF52-1F (Culture collection of Biogaia AB, Raleigh    N.C., USA)-   8. L. salivarius LS 1 (isolated from the LS 1 tablet by Frente Ltd.    Japan)-   9. L. rhamnosus GG (ATCC 53103)

In this study the above listed Lactobacillus strains are chosen to beevaluated using the selection criteria of inhibition of S. mutans andadhesion to mucin according to the invention. The methods set forth inExample 1 are used. The strains that were selected to be the mostsuitable strains are based on the combination of the S. mutansinhibiting effect and the adhesion of cells of the Lactobacillus strainto mucin. TABLE 1 Inhibition of S. mutans of Lactobacillus strains aswell as adhesion score to mucin according to the described assays. (0 =no binding, 3 = high binding, S = selected) CFU/g CFU/g CFU/g CFU/gMucin survival survival survival survival binding S. mutans S. mutans S.mutans S. mutans score Strain ratio 10:1 ratio 3:1 ratio 1:1 ratio 1:3(0-3) Selection L. reuteri 2.0E+08 8.0E+07 6.0E+07 9.0E+06 2 — SD2112 L.reuteri 1.0E+08 2.0E+07 7.0E+06 3.0E+05 3 S DSM 20016 L. reuteri 1.0E+087.0E+07 5.0E+07 9.0E+06 3 — MM2-3 L. reuteri 1.0E+08 1.0E+07 7.0E+059.0E+04 3 S CF2-7F L. reuteri 2.0E+08 2.0E+07 4.0E+06 1.0E+05 2 S MF2-3L. reuteri 9.0E+07 8.0E+07 7.0E+07 3.0E+07 1 — MF14-C L. reuteri 1.0E+088.0E+07 7.0E+07 3.0E+07 1 — MF52-1F L. salivarius 1.0E+08 8.0E+087.0E+09 3.0E+09 0-1 — LS1 L. rhamnosus 2.0E+08 8.0E+07 7.0E+07 3.0E+070-1 — GG

EXAMPLE 3 Confirmation of Efficacy of Selected Lactobacillus Strains

The effects of milk fermented with added evaluated Lactobacillus strains(test Yogurt) according to the list of Example 2, and placebo fermentedmilk (Placebo Yogurt) on oral carriage of mutans streptococci isstudied. The test yogurts are:

-   Test yogurt 1 . . . with L. reuteri SD2112-   Test yogurt 2 . . . with L. reuteri DSM 20016-   Test yogurt 3 . . . with L. reuteri MM2-3-   Test yogurt 4 . . . with L. reuteri CF2-7F-   Test yogurt 5 . . . with L. reuteri MF2-3-   Test yogurt 6 . . . with L. reuteri MF14-C-   Test yogurt 7 . . . with L. reuteri MF52-1F-   Test yogurt 8 . . . with L. salivarius LS 1-   Test yogurt 9 . . . with L. rhamnosus GG-   Test yogurt 10 Placebo, see below

200 healthy female subjects (age; 20 plus or minus 2 year) are dividedinto 10 groups. All subjects shall not have any active caries lesions,symptom of either gingivitis or periodontal disease. Subjects in thefirst group eat a cup (95 g) of Placebo Yogurt at lunch time(12:00-1300) every day, for two weeks and then eat a cup of Test Yogurtno. 1, at lunch time for another two weeks. Subjects in the second groupeat a cup (95 g) of Placebo Yogurt at lunch time (12:00-1300) every day,for two weeks and then eat a cup of Test Yogurt no. 2, at lunch time aday, for another two weeks, and so on for all 9 first groups. Test groupno. 10 eat Placebo Yogurt for both periods. Before and after the eatingof each kind of yogurt, the levels of oral carriage of mutansstreptococci is determined, as follows. Approximately 5 ml ofunstimulated whole saliva is collected in a container on ice at15:00-16:00. Then the oral carriage of mutans streptococci is determinedby conventional viable counts. Placebo Yogurt are comprised of L.bulgaris and S. thermophilus, which are widely employed in fermentedmilk products, the placebo yogurt are heated to 80° C. for 10 min tokill microorganisms and prepared as Test yogurt no 10. Both the subjectsand investigators are unaware of which yogurt contained which testorganism throughout the study. The use of other products containingLactobacillus or other pharmaceutical lactic acid bacteria is forbiddenfor one week prior to and throughout the intervention. The data isanalyzed statistically. TABLE 2 Result from In vivo inhibition of S.mutans by lactobacilli strains, data in log (CFU of oral streptococci)Yogurt After After and Strain Start Placebo Test Test yogurt 1: 5.0E+056.0E+05 1.0E+05 L. reuteri SD2112 Test yogurt 2: 3.0E+05 2.0E+05 4.0E+04L. reuteri DSM 20016 Test yogurt 3: 2.0E+05 4.0E+05 1.0E+05 L. reuteriMM2-3 Test yogurt 4: 1.0E+05 7.0E+05 9.0E+02 L. reuteri CF2-7F Testyogurt 5: 9.0E+04 7.0E+05 2.0E+03 L. reuteri MF2-3 Test yogurt 6:1.0E+03 4.0E+05 1.0E+05 L. reuteri MF14-C Test yogurt 7: 3.0E+04 7.0E+052.0E+05 L. reuteri MF52-1F Test yogurt 8: 4.0E+05 7.0E+05 1.0E+06 L.salivarius LS1 Test yogurt 9: 8.0E+04 4.0E+05 1.0E+05 L. rhamnosus GGTest yogurt 10: 7.0E+05 9.0E+05 1.0E+06 PLACEBO

EXAMPLE 4 Manufacturing of Products Containing Selected Strain

In this example, L. reuteri CF2-7F (ATTC number to be added whenassigned), was selected, using the methods above for S. mutansinhibition and mucin binding, in order to add the strain to a standardyogurt. The L. reuteri strain was grown and lyophilized, using standardmethods for growing Lactobacillus in the dairy industry. This culturewas then added to previously fermented milk, using traditional yogurtcultures, at a level of 10⁷ CFU/gram of yogurt, and the yogurt was usedby humans as a way to prevent caries.

While certain representative embodiments have been set forth herein,those skilled in the art will readily appreciate that modifications canbe made without departing from the spirit or scope of the invention.

1. A biologically pure culture of Lactobacillus reuteri strain CF2-7F(ATTC PTA-4965),
 2. A biologically pure culture of Lactobacillus reuteristrain MF2-3 (ATTC PTA-4964)
 3. A biologically pure culture ofLactobacillus selected for its ability of reducing the number ofStreptococcus mutans in the mouth for prophylaxis or treatment of dentalcaries in mammals using a selection assay of inhibiting activity incombination with good binding to oral mucins.
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